Liquid Handling Workstation
Zusammenfassung der Projektergebnisse
The liquid-handling workstation supported many projects that relied on automated processing of DNA, RNA or cells. The liquid handling workstation is equipped to handle 96- and 384-well plates in high-throughput workflows, enabling for example processes such as generation of genome-wide RNAi libraries, aliquotting of reagents in high-density tissue culture plates, transfection of nucleic acids into cells and general handling of eukaryotic cells. We have created workflows and software for the automated and autonomous performance of experiments. As such, the liquid handling platform was used in high-throughput RNAi interference screenings to analyze cellular signaling pathways. We created a novel genome-wide library of RNAi reagents that covered almost every gene in the Drosophila genome with two independent double-stranded RNAs. The liquid handling workstation was also employed in experiments to systematically analyze genetic interactions by RNAi in Drosophila cells. For example, we tested pairwise interactions between 93 genes encoding for kinases, phosphatases and adaptor proteins that regulate cell growth, proliferation and cell morphology. These included annotated components of RAS/MAPK, JNK and p38 pathways and all annotated protein and lipid phosphatases expressed in Drosophila S2 cells. The perturbed cells were fixed and stained for cellular markers and the phenotypic outcomes subsequently imaged by high-throughput microscopy. In these experiments we assessed the combined phenotypes of all pairwise RNAi treatments through independent measurements, including all possible combinations of two RNAi reagents for both target genes. We found that the phenotypic measurements were highly reproducible across replicate experiments and the two-fold coverage of each gene with two independent RNAi reagents allowed us to identify unspecific effects. Overall, we observed several hundred genetic interactions covering multiple phenotypes. We demonstrated that such a strategy could elucidate both generic and phenotype-specific genetic interactions. These studies also revealed novel modulators of RAS/MAPK signaling which were further studied by biochemical approaches in cells and through genetic analysis in vivo. We have now significantly expanded the genetic interaction network in Drosophila as well as in human cells. The largescale genetic interaction mapping by RNAi based on robust experimental designs relied on precise and scalable automation of liquid handling processes.
Projektbezogene Publikationen (Auswahl)
- Mapping of signaling networks through synthetic genetic interaction analysis by RNAi. Nature Methods 8:341-6
Horn, T., Sandmann, T., Fischer, B., Axelsson, E., Huber, W., Boutros, M.
- Extracting quantitative genetic interaction phenotypes from matrix combinatorial RNAi. BMC Bioinformatics 12:342
Axelsson, E., Sandmann, T., Horn, T., Boutros, M., Huber, W., Fischer, B.
- Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells. Nature Communications 4:2610
Voloshanenko, O., Erdmann, G., Dubash, T.D., Augustin, I., Metzig, M., Moffa, G., Hundsrucker, C., Kerr, G., Sandmann, T., Anchang, B., Demir, K., Boehm, C., Leible, S., Ball, C.R., Glimm, H., Spang, R., Boutros, M.
